Dedifferentiated, programmable stem cells of monocytic origin, and their production and use

ABSTRACT

The invention relates to the production of adult dedifferentiated, programmable stem cells from human monocytes by cultivation of monocytes in a culture medium which contains M-CSF and IL-3. The invention further relates to pharmaceutical preparations, which contain the dedifferentiated, programmable stem cells and the use of these stem cells for the production of target cells and target tissue.

DESCRIPTION

[0001] The invention relates to adult dedifferentiated programmable stemcells derived from human monocytes, as well as their production and usefor the production of body cells and tissues. According to aparticularly preferred embodiment of the invention these cells areautologous human stem cells, i.e. the cell of monocytic origin comesfrom the patient who is to be treated with the stem cell produced fromthe original cell and/or with the body cells produced from this stemcell.

[0002] In the state of the art, the term “stem cells” designates cellswhich (a) have the capability of self-renewal and (b) the capability toform at least one and often a number of specialised cell types due totheir asymmetrical division capability (cf. Donovan, P. J., Gearhart,J., Nature 414: 92-97 (2001)). The term “pluripotent” designates stemcells, which can essentially be differentiated into all possible celltypes of the human and animal body. Such stem cells have hitherto onlybeen obtainable from embryonic tissue or embryonic carcinoma (testiculartumour) (cf. Donovan, P. J., Gearhart, J., loc cit). The use ofembryonic stem cells has been the subject of extensive publicdiscussion, especially in Germany, and is regarded as extremelyproblematical. Besides the ethical and legal problems connected withembryonic stem cells, the therapeutic use of such cells also comes upagainst difficulties. By nature, embryonic stem cells are obtained fromdonor organisms, which are heterologous vis-à-vis the potentialrecipients of differentiated cells or tissue (hereafter referred to assomatic target cells or target tissue) developed from these cells. It istherefore to be expected, that such target cells will trigger animmediate immunological response in the potential recipients in the formof rejection.

[0003] Stem cells can be also isolated from different tissues of adult,i.e. from differentiated individuals. Such stem cells are referred to inthe state of the art as “multipotent adult stem cells”. In the body theyplay a role in tissue regeneration and homoeostasis. The essentialdifference between embryonic pluripotent stem cells and adultmultipotent stem cells lies in the number of differentiated tissues,which can be obtained from the respective cells. Presumably, this is dueto the fact that pluripotent stem cells come from sperm cells, or fromcells which can produce sperm, whilst adult multipotent stem cells comefrom the body or soma of adult individuals (cf. Donovan, P. J.,Gearhart, J., loc cit, Page 94), which are not capable of spermproduction.

[0004] The actual problems relating to the obtaining and use of adultstem cells however lie in the rarity of these cells. Thus, in the bonemarrow, stem cells are present only in the ratio of 1:10,000, in theperipheral blood of 1:250,000 and in the liver in the ratio of1:100,000. Obtaining such stem cells is therefore very expensive andstressful for the patient. In addition the generation of large cellquantities, as required for clinical therapy, has scarcely been possiblehitherto at reasonable expense.

[0005] This is contrasted by a constantly increasing need forpossibilities for treatment of destroyed tissue in the form of “tissueengineering” or as cell therapy, within the framework of which skin-,muscle-, heart muscle-, liver-, islet-, nerve-, neurone-, bone-,cartilage-, endothelium- and fat cells etc. are to be replaced.

[0006] In this connection, the foreseeable development of the age anddisease profile of the population in the western world is decisive,leading to the expectation of a drastic turning point in the next 10years in the health and care sector of the western European population,including the USA and Canada. In the Federal Republic of Germany alone,the demographic development suggests a 21%-growth in population in the45-64 year-old age group by 2015, and a 26%-growth in the over-65 agegroup. This is bound to result in a change in patient structure and inthe spectrum of diseases requiring treatment. Predictably, diseases ofthe cardio-circulatory system (high pressure, myocardial infarction),vascular diseases due to arteriosclerosis and metabolic diseases,metabolic diseases such an diabetes mellitus, diseases at livermetabolism, kidney diseases as well as diseases of the skeletal systemcaused by age-related degeneration, and degenerative diseases of thecerebrum caused by neuronal and glial cell losses will increase andrequire innovative treatment concepts.

[0007] These facts explain the immense national and internationalresearch and development efforts by the specialists involved, to obtainstem cells which can be programmed into differentiated cells typical oftissue (liver, bone, cartilage, muscle, skin etc.).

[0008] The problem underlying the invention therefore resides in makingavailable adult stem cells, the generation of which gives rise to noethical and/or legal problems, which are rapidly available for theplanned therapeutic use in the quantities required for this, and atjustifiable production costs, and which, when used as “cellulartherapeutics” give rise to no side effects—or none worth mentioning—interms of cellular rejection and induction of tumours, particularlymalignant tumours, in the patient in question.

[0009] According to the invention this problem is solved by theproduction of dedifferentiated programmable cells from human monocyteswhich, for the purposes of the invention, are referred to hereafter as“stem cells”. The term “dedifferentiation” is familiar to the personskilled in the relevant art, cf. for Weissman I. L., Cell 100: 157-168,FIG. 4, (2000). It signifies the regression of an adult, alreadyspecialised (differentiated) body cell to the status of a stem cell,i.e. of a cell, which in turn can be transferred (programmed) into anumber of cell types. Surprisingly, it has been demonstrated that theprocess according to the invention leads to the dedifferentiation ofmonocytes. The stem cells produced in this way can be transformed(programmed) into a large number of different target cells/targettissue, cf. examples. The stem cells according to the invention express,in addition to the CD14 surface antigen characteristic of differentiatedmonocytes, at least one, preferably two or three, of the typicalpluripotency markers CD90, CD117, CD123 and CD135. In a particularlypreferred manner, the stem cells produced according to the inventionexpress the CD14 surface antigen as well as the four pluripotencymarkers CD90, CD117, CD123 and CD135, cf. Example 2, Table 1. In thisway, for the first time adult stem cells are made available, which canwithin a short time be reprogrammed into preferably autologous tissues.

[0010] The generation of the stem cells according to the invention iscompletely harmless to the patient and—in the case of autologoususe—comparable to own blood donation. The quantity of stem cells (10⁸ to10⁹ cells) required for the usual therapy options (see, above) can bemade available cost-effectively within 10 to 14 days after the blood istaken. In addition the cell product provided for the therapy, in thecase of autologous use, does not give rise to any immunological problemin terms of cell rejection, as cells and recipient are preferablygenetically identical.

[0011] The stem cells according to the invention have also proved to berisk-free in animal experimentation and in culture with regard to givingrise to malignancy, a result which is only to be expected due to thecell of monocytic origin, from which the stem cells according to theinvention derive.

[0012] The essential steps of the process according to the invention forthe production of dedifferentiated programmable stem cells of humanmonocytic origin comprise:

[0013] (a) Isolation of monocytes from human blood;

[0014] (b) Propagating the monocytes in a suitable culture vesselcontaining cell culture medium, which contains themacrophage-colony-stimulating factor (hereafter referred to as M-CSF);and

[0015] (c) Cultivating the monocytes in the presence of interleukin-3(IL-3); and

[0016] (d) Obtaining the human dedifferentiated programmable stem cells,by separating the cells from the culture medium.

[0017] According to a particularly preferred embodiment of the process,M-CSF and IL-3 are simultaneously added to the cell culture medium inStep b).

[0018] It is however also possible, initially only to add M-CSF to thecell culture medium in Step b) in order to cause the monocytes topropagate, and to add IL-3 to the cell culture medium subsequently.

[0019] Finally the process in Step b) can also be carried out in such away that the monocytes are initially propagated in a cell culture mediumcontaining only M-CSF, then the medium is separated from the cells and asecond cell culture medium is then used, which contains IL-3.

[0020] According to a preferred embodiment of the invention the culturemedium of Step b) is separated from the cells attached to the bottom ofthe culture vessel and the human, dedifferentiated, programmable stemcells are obtained by detaching the cells from the bottom and byisolating the cells.

[0021] According to a preferred embodiment of the invention the cellsare further cultivated in the presence of a sulphur compound. Thecultivation can be carried out in a separate process step which followsthe cultivation Step b) described above. It can however also be carriedout in Step b), by further adding the sulphur compound to the culturemedium, preferably already at the start of the cultivation.

[0022] The process according to the invention surprisingly leads to thededifferentiation of the monocytes, wherein the adult stem cellsresulting from the dedifferentiation, besides the CD14 surface antigentypical of the differentiated monocytes, also express at least one ormore, preferably all of the pluripotency markers CD90, CD117, CD123 andCD135 (cf. Table 1). The expression of the respective markers (surfaceantigens) can be proved by means of commercially available antibodieswith specificity against the respective antigens to be detected, usingstandard immuno assay procedures, cf. Example 2.

[0023] As the cells, during the propagation and dedifferentiationprocess, adhere to the bottom of the respective culture vessel, it isnecessary to separate the cells from the culture medium from Step b) andto detach them from the bottom after completion of thededifferentiation. According to a preferred embodiment of the inventionthe cell culture supernatant is discarded before the detaching of thecells adhering to the bottom and subsequently, the adhering cells arepreferably rinsed with fresh culture medium. Following the rinsing,fresh culture medium is again added to the cells adhering to the bottom,and the step of releasing the cells from the bottom then follows (cf.Example 13).

[0024] According to a preferred embodiment the cells are brought intocontact with a biologically well-tolerated organic solvent, at the endof Step c) and before Step d). The biologically well-tolerated organicsolvent can be an alcohol with 1-4 carbon atoms, the use of ethanolbeing preferred.

[0025] In a further embodiment, at the end of Step c) and before Step d)the cells are brought into contact with the vapour phase of thebiologically well-tolerated organic solvent.

[0026] The detaching can moreover also be carried out mechanically,however, an enzymatic detaching process is preferred, for example withtrypsin.

[0027] The dedifferentiated programmable stem cells obtained in thisway, floating freely in the medium, can either be directly transferredto the reprogramming process, or kept in the culture medium for a fewdays; in the latter case, a cytokine or LIF (leukaemia inhibitoryfactor) is preferably added to the medium, in order to avoid prematureloss of the programmability (cf. Donovan, P. J., Gearhart, J., loc.cit., Page 94). Finally the cells can be deep-frozen for storagepurposes without loss of programmability.

[0028] The stem cells according to the invention differ from thepluripotent stem cells of embryonic origin known hitherto and from theknown adult stem cells from different tissues, in that besides themembrane-associated monocyte-specific CD14 surface antigen, they carryat least one pluripotency marker from the group consisting of CD90,CD117, CD123 and CD135 on their surface.

[0029] The stem cells produced using the process according to theinvention can be reprogrammed into any body cells. Processes forreprogramming stem cells are known in the state of the art, cf. forexample Weissman I. L., Science 287: 1442-1446 (2000) and Insight ReviewArticles Nature 414: 92-131 (2001), and the handbook “Methods of TissueEngineering”, Eds. Atala, A., Lanza, R. P., Academic Press, ISBN0-12-436636-8; Library of Congress Catalog Card No. 200188747.

[0030] The differentiated isolated somatic target cells and/or thetarget tissue obtained by reprogramming of the stem cells according tothe invention moreover carry the membrane-associated CD14differentiation marker of the monocytes. As shown in Example 11,hepatocytes which are derived from the stem cells according to theinvention, express the CD14 surface marker which is typical ofmonocytes, whilst at the same time they produce the protein albumin,which is typical of hepatocytes. The hepatocytes derived from the stemcells according to the invention can therefore be distinguished fromnatural hepatocytes. In the same way, the membrane-associated CD14surface marker was detected on insulin-producing cells, which werederived from the stem cells according to the invention (Example 9).

[0031] In one embodiment of the invention the dedifferentiated,programmable stem cells are used for the in-vitro production of targetcells and target tissue (cf. Examples). Therefore, differentiated,isolated tissue cells, which are obtained by differentiation(reprogramming) of the stem cells according to the invention, and whichcarry the membrane-associated CD14 surface antigen, are also subject ofthe present invention.

[0032] The stem cells according to the invention are preferably simplyand reliably differentiated in vitro into desired target cells, such asfor example adipocytes (cf. Example 6), neurons and glia cells (cf.Example 3), endothelial cells (cf. Example 5), keratinocytes (cf.Example 8), hepatocytes (cf. Example 7) and islet cells (islet ofLangerhans, cf. Example 9), by growing the stem cells in a medium whichcontains the supernatant of the culture medium, in which the respectivetarget cells and/or fragments thereof have been incubated (cf. Examples6 to 8). This supernatant is referred to hereafter as“target-cell-conditioned medium”.

[0033] For the differentiation (reprogramming) of the dedifferentiatedstem cells according to the invention the following procedure cantherefore be followed, in which:

[0034] a) tissue which contains or consists of the desired target cellsis crushed;

[0035] b) the tissue cells (target cells) and/or fragments of these areobtained;

[0036] c) the target cells and/or fragments of these are incubated in asuitable culture medium;

[0037] d) the culture medium supernatant is collected during and afterthe incubation as target-cell-conditioned medium; and

[0038] e) for the reprogramming/differentiation of dedifferentiated stemcells into the desired target cells or target tissue, the stem cells aregrown in the presence of the target-cell-conditioned medium.

[0039] Standard cell culture media can be used as culture medium (cf.Examples). The media preferably contain growth factors, such as forexample the epidermal growth factor.

[0040] The incubation of the target cells and/or fragments of these(“cell pellet”) can be carried out over 5 to 15, preferably 10 days. Thesupernatant, i.e. the target-cell-conditioned medium is preferablyremoved in each case after 2 to 4 days and replaced by fresh medium. Thesupernatants thus obtained can be filtered under sterile conditionsseparately or pooled and stored at approximately −20° C. or useddirectly for the programming of stem cells. As shown above, theprogramming of the stem cells into the desired target cells is carriedout by growing stem cells in the presence of the medium conditioned withthe respective target cells (cf. Examples). The growth medium preferablyadditionally contains a target-cell-specific growth factor, such as forexample the “hepatocyte growth factor” or the “keratinocyte growthfactor” (cf. Examples).

[0041] In one embodiment of the invention the dedifferentiated,programmable stem cells according to the invention are used per se forthe production of a pharmaceutical composition for the in-vivoproduction of target cells and target tissue.

[0042] Such pharmaceutical preparations can contain the stem cellsaccording to the invention suspended in a physiologically well-toleratedmedium. Suitable media are for example PBS (phosphate buffered saline)or physiological saline with 20% human albumin solution and the like.

[0043] These pharmaceutical preparations contain vital dedifferentiated,programmable stem cells according to the invention, which have on theirsurface the CD14 surface marker and at least one more of the multipotentstem cell markers CD90, CD117, CD123 and/or CD135, in a quantity of atleast 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50%, preferably 60 or70%, particularly preferably 80 or 90% and extremely preferably 100%,relative to the total number of the cells present in the preparation,and optionally further pharmaceutically well-tolerated adjuvants and/orcarrier substances.

[0044] Stem cell preparations can contain vital dedifferentiated,programmable stem cells according to the invention, which have on theirsurface the CD14 surface marker and at least one more of the pluripotentstem cell markers CD90, CD117, CD123 and/or CD135, in a quantity of atleast 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58 or 59%, preferably at least 60%, relative to the total numberof the cells present in the preparation; cell suspensions in a cellculture- or transport medium well-tolerated by cells, such as e.g. PBSor RPMI etc., or deep-frozen cell preparations in a suitable storagemedium, such as e.g. RPMI with 50% human albumin solution and 10% DMSOare preferred.

[0045] The number of vital cells and hence the proportion of these inthe compositions referred to above, can be determined optically by useof the “Trypan blue dye exclusion technique”, as vital cells can beoptically distinguished from non-vital cells, using this dye.

[0046] As a rule, it will be irrelevant for clinical use, if some of thecells present in the pharmaceutical preparation do not fulfil thecriteria of dedifferentiated, programmable stem cells according to theinvention, provided that a sufficient number of functional stem cells ispresent. It is however also possible to eliminate non-dedifferentiatedcells by means of processes known in the state of the art on the basisof surface markers typical of the dedifferentiated cells according tothe invention in such preparations, so that these contain the desiredcells in essentially pure form. One example of a suitable process is“Immuno magnetic bead sorting”, cf. Romani et al., J. Immunol. Methods196: 137-151 (1996).

[0047] Stem cells further have the capability, of spontaneouslydifferentiating in vivo by direct contact with a cell group of aspecific cell type into cells of this type. Processes for tissueproduction using cells which can be redifferentiated (“tissueengineering”) are known in the state of the art. For example Wang, X. etal. (“Liver repopulation and correction of metabolic liver disease bytransplanted adult mouse pancreatic cells” Am. J. Pathol. 158 (2):571-579 (2001)), have shown that even certain adult cells of thepancreas in mice are able to transform, in FAH— (fumaroylacetoacetatehydrolase)-deficient mice, into hepatocytes, which can fully compensatefor the metabolic defect in these animals. A further example is theexperiments of Lagasse et al., “Purified hematopoietic stem cells candifferentiate into hepatocytes in vivo”, Nature Medicine, 6 (11):1229-1234 (2000). The authors have shown that hematopoietic stem cellsfrom bone marrow were able, after in-vivo transfer into FAH-deficientmice, to transform into hepatocytes, which could then compensate for themetabolic defect; see also the review by Grompe M., “Therapeutic LiverRepopulation for the Treatment of Metabolic Liver Diseases” Hum. Cell,12: 171-180 (1999).

[0048] Particularly preferable forms of application for the in-vivodifferentiation of the dedifferentiated stem cells according to theinvention are injection, infusion or implantation of the stem cells intoone specific cell association in the body, in order to allow for thestem cells to differentiate there, by direct contact with the cellassociation, into cells of this cell type. For injection or infusion thecells can be administered in PBS (phosphate buffered saline).

[0049] Preferred examples of the relevant indications in this connectionare: cirrhosis of the liver, pancreatic insufficiency, acute or chronickidney failure, hormonal under-functioning, cardiac infarction,pulmonary embolism, stroke and skin damage.

[0050] Therefore preferred embodiments of the invention are the use ofthe dedifferentiated, programmable stem cells for the production ofdifferent pharmaceutical compositions for the treatment of cirrhosis ofthe liver, pancreatic insufficiency, acute or chronic kidney failure,hormonal under-functioning, cardiac infarction, pulmonary embolism,stroke and skin damage.

[0051] For the therapeutic use of the target cells obtainable from thestem cells according to the invention, a number of concepts areavailable (see above Science 287: 1442-1446 (2000) and Nature 414:92-131 (2001)).

[0052] A further preferred application concerns the injection of thededifferentiated stem cells according to the invention into theperitoneum, so that they differentiate there, due to the influence ofthe cells surrounding them, into peritoneal cells. In the case ofperitoneal dialysis of patients with kidney insufficiency, these cellscan take over a kidney function via their semi-permeable membrane andgive off kidney dependent waste substances into the peritoneum fromwhere these are removed via the dialysate.

[0053] Therefore, also the differentiated, isolated, somatic targetcells and/or target tissue, which are obtained by reprogramming of thestem cells and are characterised by the membrane-associated CD14 antigenare subject of the invention. These somatic target cells and/or targettissue preferably contain adipocytes, neurons and glia cells,endothelial cells, keratinocytes, hepatocytes and islet cells.

[0054] However the cells can also be introduced directly into the organto be reconstituted. The introduction can be carried out via matrixconstructions which are coated with corresponding differentiated cellsor cells capable of differentiation. The matrix constructions are as arule biodegradable, so that they disappear out of the body while thenewly introduced cells grow together with the cells present. From thispoint of view, for example cellular, preferably autologous transplantsin the form of islet cells, hepatocytes, fat cells, skin cells, muscles,cardiac muscles, nerves, bones, endocrine cells etc. come underconsideration for restitution for example after partial surgicalresection of an organ, for repair for example after trauma or forsupportive use, for example in the case of lacking or insufficient organfunction.

[0055] The stem cells according to the invention and target cellsobtained from them can further be used to coat implantable materials, inorder to increase biocompatibility. Therefore, also implantablematerials, which are coated with the dedifferentiated, programmable stemcells or the somatic target cells and/or target tissue are subject ofthe invention. According to one embodiment of the invention theseimplantable materials are prostheses. In particularly preferredembodiments these prostheses are cardiac valves, vessel prostheses,bone- and joint prostheses.

[0056] The implantable materials can also be artificial and/orbiological carrier materials, which contain the dedifferentiated,programmable stem cells or target cells. In this regard, the carriermaterials can be bags or chambers for insertion into the human body.

[0057] In one embodiment of the invention such a bag, containing isletcells, which are differentiated somatic cells according to theinvention, is used for the production of a pharmaceutical construct foruse as an artificial islet cell port chamber for the supply of insulin.

[0058] According to a further embodiment of the invention, a bag orchamber containing adipocytes, which are differentiated somatic cellsaccording to the invention, is used for the production of an artificialpolymer filled with adipocytes as a pharmaceutical construct for breastconstruction after surgery and in the case of further indications ofplastic and/or cosmetic correction.

[0059] Moreover, semi-permeable port chamber systems, containingendocrine cells of very widely varying provenance, can be used in vivofor the treatment of endocrine, metabolic or haemostatic disorders.Examples of such endocrine cells are cells which produce thyroxine,steroids, ADH, aldosterone, melatonin, serotonin, adrenalin,noradrenalin, TSH, LH, FSH, leptin, cholecystokinin, gastrin, insulin,glucagon, or clotting factors.

[0060] Therefore, also implantable materials, which are semi-permeableport chamber systems, containing differentiated isolated somatic targetcells are subject of the invention. These semi-permeable chamber systemsare used in different embodiments of the invention for the production ofa pharmaceutical construct for the in-vivo treatment of endocrine,metabolic or haemostatic disorders.

[0061] The target cells obtained from the stem cells according to theinvention can in addition be used as cell cultures in bioreactorsoutside the body, for example in order to carry out detoxificationreactions. This form of use is particularly relevant in the case ofacute conditions, for example in the case of acute liver failure as ahepatocyte-bioreactor.

[0062] The production of the constructs described above and conductingthe corresponding therapeutic process have already been described manytimes in the state of the art, compare for example the review by Lalan,S., et al. “Tissue engineering and its potential impact on surgery”World J. Surg. 25: 1458-1466 (2001); Nasseri, B. A., et al. “Tissueengineering: an evolving 21st-century science to provide replacement forreconstruction and transplantation” Surgery 130: 781-784 (2001) andFuchs, J. R., et al., “Tissue engineering: a 21st century solution tosurgical reconstruction” Ann. Thorac. Surg. 72: 577-591 (2001).

[0063] Finally, the pluripotent stem cells according to the inventionopen up a broad field for transgenic modification and therapy. Accordingto a preferred embodiment of the invention the dedifferentiatedprogrammable stem cells per se or somatic target cells and/or targettissue finally differentiated from these, are transfected with one ormore genes. In this way, one or more genes which are required tomaintain the metabolism of certain organs, such as for example livers orkidneys, are restored and/or supported or reintroduced. For example,stem cells or hepatocytes derived from these can be transfected with theFAH (fumaroylacetoacetate hydrolase) gene. In the FAH-deficient mousemodel the intrasplenic injection of 1000 FAH-positive donor hepatocyteswas sufficient to completely repopularise the liver after 6 to 8 weeksand fully compensate for the metabolic defect leading to cirrhosis ofthe liver (cf. Grompe, M., et al., Nat. Genet. 12: 266 ff. (1996)).

[0064] Correspondingly, by transfection of the stem cells or therespective target cells obtained from the stem cells by programming (forexample hematopoietic cells, hepatocytes, ovary cells, muscle cells,nerve cells, neurons, glia cells, cartilage or bones cells, etc.) with“Multi-Drug-Resistance-genes” extended radical chemotherapy can be madepossible in the case of malignant diseases by correspondinghematopoietic reconstitution or radiation resistance can be produced.

[0065] The invention is explained in detail as follows:

[0066] The starting material for the process according to the inventionis monocytes from human blood. These are preferably autologousmonocytes, i.e. monocytes, which originate from the blood of the patientto be treated with the stem cells according to the invention or thetarget cells produced from these.

[0067] To obtain the monocytes the blood can first, after standardtreatment with an anticoagulant in a known manner, preferably bycentrifugation, be separated into plasma and into white and red bloodcells. After the centrifugation the plasma is to be found in thesupernatant; below this lies a layer which contains the totality of thewhite blood cells. This layer is also referred to as “buffy coat”. Belowthis lies the phase containing red blood cells (haematocrit).

[0068] The “buffy coat” layer is then isolated and separated to obtainthe monocytes for example by centrifuging using a known process.According to a preferred process variant the “buffy coat” layer iscoated onto a lymphocyte separation medium (e.g. Ficoll Hypaque) andcentrifuged. By further centrifuging and rinsing, the monocyte fractionis obtained from the blood (cf. Example 1).

[0069] Examples of alternative processes for obtaining the monocytesfrom complete blood are “Fluorescence-Activated Cell Sorting” (FACS),“Immunomagnetic Bead Sorting” (cf. Romani et al., J. Immunol. Methods196: 137-151 (1996)) and “Magnetic-Activated Cell Sorting” (MACS) or theso called “Rosetting process” (cf. Gmelig-Meyling, F., et al.,“Simplified procedure for the separation of human T and non-T cells” VoxSang. 33: 5-8 (1977)).

[0070] According to the invention, monocytes can be obtained from anyisolated human blood, and the blood can also originate from organs suchas the spleen, lymph nodes or bone marrow. Obtaining monocytes fromorgans is considered especially when the separation of the monocytesfrom human blood, e.g. in the case of anaemia or leukaemia, is notpossible, or not in sufficient quantities, and in the case of allogenicuse, if, within the framework of multi-organ removal, the spleen isavailable as a source for isolation of monocytes.

[0071] For the production of a sufficient quantity of stem cellsaccording to the invention it is first necessary to propagate themonocytes. For this purpose, growth media suitable for monocytes can beused, wherein, according to the invention said medium containes M-CSF(macrophage colony stimulating factor). M-CSF (also referred to asCSF-1) is produced by monocytes, fibroblasts and endothelial cells. Theconcentration of M-CSF in the culture medium can amount to 2 to 20 μg/lmedium, preferably 4 to 6 μg/l and in a particularly prefered manner 5μg/l.

[0072] On the monocytes M-CSF binds to the specific c-Fms receptor (alsoreferred to as CSF-1R), which is exclusively present on the surface ofmonocytes and which only binds M-CSF (Sherr C. J., et al., Cell 41 (3):665-676 (1985)). As the specific interaction between M-CSF and thereceptor induces the division of the monocytes, the medium, in which themonocytes are cultivated contains M-CSF or an analogue thereof, whichcan bind to the receptor and activate it. Other growth factors such asGM-CSF (granulocyte-monocyte colony stimulating factor) and G-CSF(granulocyte colony stimulating factor) are unsuitable, as, due to thelack of affinity to the c-Fms receptor, they are not capable of inducingmonocyte division.

[0073] In a particularly preferred embodiment of the process M-CSF andIL-3 are simultaneously added to the cell culture medium in Step b) ofthe process. The concentration of IL-3 in the medium may amount to 0.2to 1 μg/l, preferably 0.3 to 0.5 μg/l and in a particularly preferredmanner 0.4 μg IL-3/1.

[0074] It is however also possible, to add initially only M-CSF to thecell culture medium in Step b) and add IL-3 only thereafter.

[0075] In a further embodiment the culture vessel initially containscell culture medium which contains only M-CSF, which after theseparation of the cells is then replaced by a second cell culturemedium, which contains IL-3.

[0076] According to a preferred embodiment of the invention the cells inStep b) of the process are additionally cultivated in the presence of asulphur compound, e.g. a mercapto compound, in which at least onehydrocarbon group is bonded to the sulphur, and said hydrocarbongroup(s) may be substituted with one or more functional groups. Mercaptocompounds are defined as compounds which have at least one mercaptogroup (—SH), which is bonded to a hydrocarbon group. By the additionaluse of such a sulphur compound, the number of the stem cells obtained bydedifferentiation of the cells of monocytic origin, which express one ormore of the stem cell markers CD90, CD117, CD123 and CD135, can beincreased.

[0077] The functional group(s) is/are preferably hydroxyl- and/or aminegroups. In a particularly preferred embodiment, the sulphur compound is2-mercaptoethanol. According to a further preferred embodiment thesulphur compound is dimethylsulfoxide (DMSO).

[0078] The quantity of the sulphur compound used can range fromapproximately 4 to approximately 200 μmol/l relative to the sulphur.Approximately 100 μmol/l is preferred.

[0079] When 2-mercaptoethanol is used, the culture medium should containapproximately 3 μl to approximately 13 μl, preferably approximately 7 μl2-mercaptoethanol/l.

[0080] The treatment with IL-3 and optionally with the sulphur compoundcan be carried out simultaneously with or following the propagation ofthe monocytes by cultivation with M-CSF, simultaneous propagation andtreatment with IL-3 and optionally a sulphur compound being prefered.Propagation and dedifferentiation should, taken together, last no morethan 10 days, and the treatment with IL-3 and optionally with thesulphur compound should be carried out over at least 3 and at most 10days, preferably 6 days.

[0081] Therefore, according to the invention, in the case of cultivationof the monocytes in a culture medium, which simultaneously containsM-CSF, IL-3 and preferably a mercapto compound, the duration ofcultivation until the detaching of the cells from the bottom of theculture vessel amounts to at least 3 and at most 10 days, preferably 5to 8 days and particularly preferably 6 days.

[0082] If in a preferred embodiment the process according to theinvention is carried out in such a way that the monocytes in Step b) areinitially propagated in a medium containing only M-CSF, the propagationin such a culture medium can take place over a period of at least 2,preferably 3 and particularly preferably 4 days with a maximum durationof 7 days, and a subsequent cultivation in the presence of IL-3 andoptionally of a mercapto compound can take place over a further 3 days.Preferably in such a case the cultivation in a medium containing onlyM-CSF will however only last a maximum of 4 days, followed by acultivation in the presence of IL-3 and optionally of a mercaptocompound over a period of 3, 4, 5 or 6 days.

[0083] To carry out the propagation and dedifferentiation jointly, asdescribed in Examples 2 and 13, the monocytes are after isolationtransferred into a medium, which contains both MCSF, and IL-3 as well aspreferably the sulphur compound, in particular mercaptoethanol or DMSO.

[0084] Due to their adhesive properties the monocytes and the stem cellsproduced from them during the process adhere to the bottom of therespective culture vessel. According to a preferred embodiment of theinvention, the culture medium is after Step c) separated from the cellsadhering to the bottom of the culture vessel and is discarded. This ispreferably followed by rinsing of the cells adhering to the bottom withculture medium, and the cells are then covered with fresh culture medium(cf. Example 13).

[0085] In this step the propagation and dedifferentiation mediumdescribed above can be used as culture medium, as well as a standardcell culture medium, for example RPMI.

[0086] According to a further prefered embodiment of the invention, thecells are brought into contact with a biologically well-toleratedorganic solvent at the end of Step c) and before Step d), in order toincrease the number of stem cells floating freely in the medium at theend of the process. The quantity of the solvent can range from 10 μl to1 ml. This is preferably an alcohol with 1-4 carbon atoms, the additionof ethanol being particularly prefered. According to a particularlypreferred embodiment the cells are brought into contact with the vapourphase of the previously defined biologically well-tolerated organicsolvent, preferably with ethanol vapour (cf. Example 2). The time forexposure to the organic solvent, particularly preferably to ethanolvapour, should amount to 4-12 hours, preferably 8-10 hours.

[0087] The process according to the invention is preferably carried outin culture vessels, the surface of which has previously been coated withfoetal calf serum (FCS) (cf. Example 2). Alternatively human AB-Serumfrom male donors can be also be used. The coating with FCS can becarried out by covering the surface of culture vessels with FCS beforeuse, and after an exposure time of a few, in particular 2 to 12 hours,and in a particularly preferable manner 7 hours, and by removing the FCSnot adhering to the surface in a suitable manner.

[0088] If treatment with organic solvent take place after Step c)optionally after exchange of the culture medium, the cells alreadybecome detached from the bottom to a certain extent in this processstep. The (further) detaching can be carried out mechanically, forexample with a fine cell scraper, spatula or tip of a pipette (cf.Example 13).

[0089] According to a preferred embodiment of the process, completedetaching is carried out by treatment with a suitable enzyme, forexample with trypsin (cf. Example 2). The cells may be exposed to thetrypsin solution (0.1 to 0.025 g/l, preferably 0.05 g/l) for 2-10minutes at 35° C. to 39° C., preferably at 37° C., in the presence ofCO₂.

[0090] The trypsin activity is then blocked by a standard method, andthe now freely floating dedifferentiated programmable stem cells can beobtained by a standard method, for example by centrifuging and in oneembodiment by suspended in a suitable cell culture at the end of Stepd). They are now available, suspended in a suitable medium, for examplein RPMI 1640 or DMEM, for immediate differentiation into the desiredtarget cells. They can however also be stored in the medium for a fewdays. In a preferred embodiment the medium contains a cytokine or LIFfactor (leukemia inhibitory factor), cf. Nature 414: 94 (2001, Donovan,P. J., Gearhardt, J., loc. cit.), if the cells are to be stored inculture for longer than approximately 48 hours as dedifferentiatedprogrammable stem cells. In a medium containing such factors stem cellscan be kept for at least 10 days as dedifferentiated programmable stemcells.

[0091] In a preferred embodiment the cells are suspended for longerstorage in a liquid medium and then deep-frozen. Protocols for the deepfreezing of living cells are known in the state of the art, cf. GriffithM., et al. “Epithelial Cell Culture, Cornea, in Methods of TissueEngineering”, Atala A., Lanza R. P., Academic Press 2002, Chapter 4,Pages 131 to 140. A preferred suspension medium for the deep freezing ofthe stem cells according to the invention is FCS-containing DMEM, cf.Example 2.

[0092] The invention is further exemplified and described below withreference to examples.

[0093] If not defined within the examples, the composition of the mediaand substances used are as follows:

[0094] 1. Penicillin/streptomycin solution:

[0095] 10,000 units of penicillin as sodium salt of penicillin G and1000 μg streptomycin as streptomycin sulphate per ml physiologicalsodium chloride solution (NaCl 0.9%)

[0096] 2. Trypsin-EDTA

[0097] 0.5 g trypsin and 0.2 g EDTA (4 Na)/l

[0098] 3. Insulin

[0099] human, recombinant, produced in E. coli, approximately 28units/mg

[0100] 4. RPMI 1640 (lx, liquid (11875)) contains L-Glutamine

[0101] RPMI (Roswell Park Memorial Institute) Media 1640 are enrichedformulations, which can be used extensively for mammalian cells. Mol.-Conc. Molarity Components weight (mg/l) (nM) Anorganic salts Calciumnitrate (Ca(NO₃)₂ 4H₂O) 236 100.00 0.424 Potassium chloride (KCl) 75400.00 5.30 Magnesium sulphate (MgSO₄) 120 48.84 0.407 Sodium chloride(NaCl) 58 6000.00 103.44 Sodium bicarbonate (NaHCO₃) 84 2000.00 23.800Sodium phosphate (Na₂HPO₄) 142 800.00 5.63 Further components Glucose180 2000.00 11.10 Glutathione, reduced 307 1.50 0.0032 Phenol red 3985.00 0.0125 Amino acids L-Arginine 174 200.00 1.10 L-Asparagine 13250.00 0.379 L-Asparaginic acid 133 20.00 0.150 L-Cysteinedihydrochloride 313 65.00 0.206 L-Glutamininc acid 147 20.00 0.136L-Glutamine 146 300.00 2.05 Glycine 75 10.00 0.133 L-Histidine 155 15.000.0967 L-Hydroxyproline 131 20.00 0.153 L-Isoleucine 131 50.00 0.382L-Leucine 131 50.00 0.382 L-Lysine hydrochloride 146 40.00 0.219L-Methionine 149 15.00 0.101 L-Phenylalanine 165 15.00 0.0909 L-Proline115 20.00 0.174 L-Serine 105 30.00 0.286 L-Threonine 119 20.00 0.168L-Tryptophan 204 5.00 0.0245 L-Tyrosine disodium, dihydrate 261 29.000.110 L-Valine 117 20.00 0.171 Vitamins Biotin 244 0.20 0.008 D-calciumpantothenate 477 0.25 0.0005 Choline chloride 140 3.00 0.0214 Folic acid441 1.00 0.0022 i-Inositol 180 35.00 0.194 Niacinamide 122 1.00 0.0081p-aminobenzoic acid (PABA) 137 1.00 0.0072 Pyridoxine HCl 206 1.000.0048 Riboflavin 376 0.20 0.0005 Thiamin HCl 337 1.00 0.0029 VitaminB12 1355 0.005 0.00000369

[0102] Reference: Moore G. E., et al., J.A.M.A. 199: 519 (1967)

[0103] 5. PBS (Dulbecco's phosphate buffered saline) cf. J. Exp. Med.98:167 (1954): Components g/l KCl 0.2 KH₂PO₄ 0.2 NaCl 8.00 Na₂PHO₄ 1.15

[0104] 6. 2-Mercaptoethanol

[0105] Quality for synthesis; Content >98%, Density 1.115 to 1.116, cf.e.g. Momo J., et al., J. Am. Chem. Soc. 73: 4961 (1951).

[0106] 7. Ficoll-Hypaque:

[0107] Lymphocyte separation medium(saccharose/epichlorohydrin-copolymerisate Mg 400,000; Density 1.077,adjusted with Sodium diatrizoate).

[0108] 8. Retinic acid:

[0109] Vitamin A acid (C₂₀H₂₈ _(O) ₂), 300 μl in 1.5 ml PBScorresponding to 1 mM. As medium for programming of neurons and gliacells use 150 μl on 10 ml medium (corresponding to 10⁻⁶ M).

[0110] 9. DMEM

[0111] Dulbecco's modified Eagle medium (high glucose) cf. Dulbecco, R.et al., Virology 8: 396 (1959); Smith, J. D. et al., Virology 12: 158(1960); Tissue Culture Standards Committee, In Vitro 6: 2 (1993)

[0112] 10. L-Glutamine

[0113] Liquid: 29.2 mg/ml

[0114] 11. Collagenase Type II:

[0115] Cf. Rodbell, M. et al., J. Biol. Chem. 239: 375 (1964).

[0116] 12. Interleukin-3 (IL-3):

[0117] Recombinant human IL-3 from E. coli (Yang Y. C. et al., Cell 47:10 (1986)); contains the 133 amino acid residues including mature IL-3and the 134 amino acid residues including the methionyl form in a ratioof approximately 1:2; calculated mol. mass approximately 17.5 kD;specific activity 1×10³ U/μg; (R&D Catalogue No. 203-IL)

[0118] 13. Macrophage-colony stimulating factor (M-CSF)

[0119] Recombinant human M-CSF from E. coli; contains as monomer (18.5kD) 135 amino acid residues including the N-terminal methionine; ispresent as a homodimer with a molar mass of 37 kD; (SIGMA Catalogue No.M 6518)

[0120] 14. Antibodies:

[0121] The antibodies used in the examples against the antigens CD14,CD31, CD90, CD117, CD123, CD135 are commercially available. They wereobtained from the following sources:

[0122] CD14: DAKO, Monoclonal Mouse Anti-Human CD14, Monocyte, CloneTUK4, Code No. M 0825, Lot 036 Edition 02.02.01;

[0123] CD31: Pharmingen International, Monoclonal Mouse Anti-Rat CD31(PECAM-1), Clone TLD-3A12, Catalogue No. 22711D, 0.5 mg;

[0124] CD90: Biozol Diagnostica, Serotec, Mouse Anti-Human CDw90, CloneNo. F15-42-1, MCAP90, Batch No. 0699;

[0125] CD117: DAKO, Monoclonal Mouse Anti-Human CD117, c-kit, Clone No.104D2, Code No. M 7140, Lot 016, Edition 04.05-0.00;

[0126] CD123: Research Diagnostics Inc., Mouse Anti-human CD123antibodies, Clone 9F5, Catalogue No. RDI-CD123-9F5;

[0127] CD135: Serotec, Mouse Anti-Human CD135, MCA1843, Clone No.BV10A4H2.

EXAMPLE 1 Separation of Monocytes from Whole Blood

[0128] To avoid blood clotting and to feed the cells, 450 ml of wholeblood in a 3-chamber bag set was mixed with 63 ml of a stabilisingsolution, which contained for each litre of H₂O, 3.27 g citric acid,26.3 g trisodium citrate, 25.5 g dextrose and 22.22 g sodiumdihydroxyphosphate. The pH-value of the solution amounted to 5.6-5.8.

[0129] “Sharp centrifugation” of this mixture was then carried out toseparate the blood components at 4000 rpm for 7 minutes at 20° C. Thisresulted in a 3-fold stratification of the corpuscular andnon-corpuscular components. By inserting the set of bags into a pressingmachine provided for this purpose, the erythrocytes were then pressedinto the lower bag, the plasma was pressed into the upper bag, and the“Buffy-coat” remained in the middle bag, and it contained approximately50 ml in volume.

[0130] The quantity of 50 ml freshly obtained “Buffy-coat” was thendivided into 2 portions of 25 ml each, each of which was then coatedwith 25 ml Ficoll-Hypaque separation medium, which had been introducedinto two 50 ml Falcon tubes beforehand.

[0131] This mixture was centrifuged without brake for 30 minutes at 2500rpm. Thereafter, erythrocytes and dead cells still present in the “Buffycoat” lay below the Ficoll phase whilst the white blood cells includingthe monocytes are separated as a white interphase on the Ficoll.

[0132] The white interphase of the monocytes was then carefully pipettedoff and was mixed with 10 ml of phosphate buffered physiological saline(PBS).

[0133] This mixture was then centrifuged with brake three times for 10minutes at 1800 rpm; the supernatant was pipetted off after eachcentrifugation and fresh PBS was filled up.

[0134] The cell sediment collected on the base of the centrifugationvessel (Falcon tube) contained the mononuclear cell fraction, i.e. themonocytes.

EXAMPLE 2 Propagation and Dedifferentiation of the Monocytes

[0135] The cultivation and propagation of the monocytes on the one handand the dedifferentiation of the cells on the other hand were carriedout in one step in nutrient medium of the following composition: RPMI1640 medium 440 ml Foetal calf serum (FCS)  50 mlPenicillin/Streptomycin solution  5 ml 2-Mercaptoethanol  5 ml (Stocksolution) Total volume 500 ml

[0136] The nutrient medium further contained 2.5 μg/500 ml of M-CSF and0.2 μg/500 ml interleukin-3 (IL-3).

[0137] The monocytes isolated in Example 1 were transferred into 5chambers of a 6-chamber well plate (30 mm diameter per well) in aquantity of approximately 10⁵ cells per chamber in each case, and filledup in each case with 2 ml of the above-mentioned nutrient medium. The6-well plate was previously filled with pure, inactivated FCS and theFCS was decanted after approximately 7 hours, in order to obtain anFCS-coated plate in this way. The cell number for the exact dose perwell was determined according to a known process, cf. Hay R. J., “CellQuantification and Characterisation” in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 55-84.

[0138] The 6-well plate was covered with its lid and stored for 6 daysin an incubator at 37° C. The cells settled to the bottom of thechambers after 24 hours. Every second day the supernatant was pipettedoff and the chambers of the 6-well plate were again each filled up with2 ml of fresh nutrient medium.

[0139] On the 6th day 2 ml of 70% ethanol was introduced into the 6-wellplate's 6th chamber which had remained free, the plate was again closedand was stored for a further 10 hours at 37° C. in the incubator.

[0140] Subsequently, 1 ml of a trypsin solution diluted 1:10 with PBSwere pipetted into each of the chambers of the well plate whichcontained cells. The closed well plate was placed for 5 minutes at 37°C. under 5% CO₂ in the incubator.

[0141] The trypsin activity was subsequently blocked by the addition of2 ml of RPMI 1640 medium to each of the wells. The total supernatant ineach of the chambers (1 ml trypsin+2 ml medium) was pipetted off, pooledin a 15 ml Falcon tube and centrifuged for 10 minutes at 1800 rpm. Thesupernatant was then discarded and the precipitate was mixed with freshRPMI 1640 medium (2 ml/10⁵ cells).

[0142] This cell suspension could be directly used for differentiationinto different target cells.

[0143] Alternatively, after centrifugation and discarding of thetrypsin-containing supernatant the cells were mixed with DMSO/FCS as afreezing medium and deep-frozen at a concentration of 10⁶/ml.

[0144] The freezing medium contained 95% FCS and 5% DMSO. In each caseapproximately 10⁶ cells were taken up in 1 ml of the medium and cooleddown in the following steps:

[0145] 30 minutes on ice;

[0146] 2 hours at −20° C. in pre-cooled Styropor boxes;

[0147] 24 hours at −80° C. in Styropor;

[0148] Storage in tubes in liquid nitrogen (N₂) at −180° C.

[0149] For immune-histochemical phenotyping of the cell population ofdedifferentiated programmable stem cells of monocytic origin, generatedaccording to the above process, in each case 10⁵ cells were taken andfixed as a cytospin preparation on slides for further histochemicalstaining (Watson, P. “A slide centrifuge; an apparatus for concentratingcells in suspension on a microscope slide.” J. Lab. Clin. Med., 68:494-501 (1966)). After this the cells could be stained using thetechnique described by Cordell, J. L., et al., (Literature, see below)with APAAP red complex. If not indicated otherwise, the added primaryantibody was diluted 1:100 with PBS, and in each case 200 μl of thisconcentration of antibodies was used. Monoclonal antibodies were used asprimary antibodies against the cell antigen epitopes listed in Table 1.FIG. 6 shows stained cytospin preparations and the corresponding proofof the stem cell markers CD90, CD117, CD123 and CD135.

[0150] Literature Relating to Staining Technique:

[0151] Cordell J. L., et al. “Immunoenzymatic labeling of monoclonalantibodies using immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32: 219-229 (1984).

[0152] Literature Relating to the Markers:

[0153] CD14

[0154] Ferrero E., Goyert S. M. “Nucleotide sequence of the geneencoding the monocyte differentiation antigen, CD14” Nucleic Acids Res.16: 4173-4173 (1988).

[0155] CD31

[0156] Newman P. J., Berndt M. C., Gorski J., White J. C. II, Lyman S.,Paddock C., Muller W. A. “PECAM-1 (CD31) cloning and relation toadhesion molecules of the immunoglobulin gene superfamily” Science 247:1219-1222 (1990).

[0157] CD90

[0158] Seki T., Spurr N., Obata F., Goyert S., Goodfellow P., Silver J.“The human thy-1 gene: structure and chromosomal location” Proc. Natl.Acad. Sci. USA 82: 6657-6661 (1985).

[0159] CD117

[0160] Yarden Y., Kuang W.-J., Yang-Feng T., Coussels L., Munemitsu S.,Dull T. J., Chen E., Schlessinger J., Francke U., Ullrich A. “Humanproto-oncogene c-kit: a new cell surface receptor tyrosine kinase for anunidentified ligand.” EMBO J. 6: 3341-3351 (1987).

[0161] CD123

[0162] Kitamura T., Sato N., Arai K., Miyajima A. “expression cloning ofthe human IL-3 receptor cDNA reveals a shared beta subunit for the humanIL-3 and GM-CSF receptors.” Cell 66: 165-1174 (1991).

[0163] CD135

[0164] Small D., Levenstein M., Kim E., Carow C., Amn S., Rockwell P.,Witte L., Burrow C., Ratajazak M. Z., Gewirtz A. M., Civin C.I.

[0165] “STK-1, the human homolog of Flk-2/Flt-3, is selectivelyexpressed in CD34+ human bone marrow cells and is involved in theproliferation of early progenitor/stem cells.” Proc. Natl. Acad. Sci.USA 91: 459-463 (1994).

Table 1

[0166] Antigen Expression of the Stem Cells According to the InventionColour Antigen reaction Stem cell marker CD90 ++ CD117 + CD123 ++CD135 + Differentiation marker CD14 (monocytes) +

[0167] Only cytospin preparations which had more than 70% vital cellswith typical stem cell morphology (cf. FIG. 6) were evaluated.

EXAMPLE 3 Production of Neurons and Glia Cells from Adult Stem Cells

[0168] The production of neurons and glia cells was carried out in petridishes with a diameter of 100 mm. To prepare the petri dishes, 5 ml ofpure inactivated foetal calf serum (FCS) was introduced into each dish,so that the bottom was covered. After 7 hours, the proportion of FCS notadhering to the bottom of the petri dish was pipetted off. Approximately10⁶ of the cells produced in accordance with Example 2 were introducedinto one of the prepared petri dishes and 10 ml of nutrient medium ofthe following composition was added: DMEM solution 440 ml Fetal calfserum (FCS)  50 ml 1-Glutamine  5 ml Penicillin (100 U/l)/Streptomycin 5 ml (100 μg/l) solution Total volume 500 ml

[0169] The nutrient medium further contained retinic acid in a quantityof 1×10⁻⁶ M/500 ml.

[0170] The reprogramming/differentiation of the stem cells used intoneurons and glia cells took place within 10 days, the medium beingchanged at intervals of approximately 3 days. After this period, thecells were mostly adhering to the bottom of the chamber and could bedetached by brief trypsinization from the bottom of the plate in amanner analogous to that previously described for the stem cells.

EXAMPLE 4 Evidence of Neuronal Precursor Cells, Neurons and Glia Cells

[0171] For the later immunohistochemical characterisation of the targetcells induced by the dedifferentiated programmable stem cells, the stemcells generated from monocytes (10⁵ cells/glass lid) were applied toglass lids (20 mm×20 mm), which were placed on the bottom of the 6-wellplates (30 mm diameter per chamber) and cultivated with the nutrientmedium (2 ml) per well plate. After the respective target cells weredifferentiated, these were fixed as follows: After removal of thenutrient medium (supernatant) the cultivated target cells were fixed bythe addition of 2 ml Methanol, which took effect over 10 minutes.Subsequently the ethanol was pipetted off, and the well plates werewashed twice with PBS (2 ml in each case). After this, the cells couldbe stained with APAAP red complex using the technique described byCordell, J. L., et al., “Immunoenzymatic labeling monoclonal antibodiesusing immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32: 219-229 (1994). Unless otherwise specified, the added primaryantibody was diluted 1:100 with PBS, in each case 200 μl of thisconcentration of antibodies were pipetted into each of the 6 wells.

[0172] Neuronal precursor cells were detected by staining the cells withthe antibody against the S100-antigen, cf. middle picture of FIG. 1(×200).

[0173] Neurons were detected by specific expression of synaptophysinMAP2 (microtubular associated protein 2) or neurofilament 68 with thecorresponding specific antibodies (primary antibody diluted 1:300 withPBS), right-hand picture of FIG. 1, ×200.

[0174] Glia cells, such as for example astrocytes, were identified bydetection of GFAP (glial fibrillary associated protein) (primaryantibody diluted 1:200 with PBS), left-hand picture of FIG. 1, ×200.

[0175] The separation of neurons and glia cells was carried out usingantibodies specific against MAP2 (neurons) or GFAP (glia cells), bymeans of MACS (Magnetic Activated Cell Sorting) according to the processas described for example in Carmiol S., “Cell Isolation and Selection”Methods of Tissue Engineering, Academic Press 2002, Chapter 2, Pages19-35.

[0176] The cell types made visible by staining are shown in FIG. 1.

EXAMPLE 5 Production of Endothelial Cells from DedifferentiatedProgrammable Adult Stem Cells of Monocytic Origin

[0177] For the cultivation of endothelial cells, Matrigel® (Beckton andDickinson, Heidelberg, DE) was used as matrix. This matrix consists offibronectin, laminin and collagens I and IV.

[0178] The frozen matrix was slowly thawed at 4° C. in a refrigeratorover a period of 12 hours. During this period its state changed, i.e.the originally solid matrix became spongy/liquid. In this state it wasintroduced into a 48-well plate (10 mm diameter per well) in such amanner, that the bottom of each of the wells was covered.

[0179] After application, the plate was kept for 30 minutes at roomtemperature, until the gel had solidified at the bottom as an adherentlayer.

[0180] Subsequently approximately 1×10² cells per well were incubated onMatrigel® with addition of the nutrient medium (as described in Example2).

[0181] After 4-5 days the first tubular cell strands appeared, whichdeveloped after 6-8 days into three-dimensional cell networks. On thecells, the endothelial markers CD31 and factor VIII could be identifiedwith the respective specific primary antibodies (200 μl, in each casediluted to 1:100 with PBS).

[0182] In an alternative process the liquefied matrix was applied to avessel-prosthesis, which was then coated with the dedifferentiatedprogrammable adult stem cells according to Example 2. Afterapproximately 6 days a lawn of endothelial cells could be identified,which coated the prosthesis in a circular manner.

[0183] The endothelial cells made visible by staining with correspondingendothelium-specific antibodies (see above) are shown in FIG. 2. In themiddle picture, the cells are shown after 5 days' incubation onMatrigel®. First tubular strands combine individual cell aggregates. Thedark-brown marked cells express CD31 antigen (×200 with yellow filter).After 8 days there is an increasing formation of three-dimensionalnetwork structures takes place (anti-CD31-antigen staining, ×200 withyellow filter). After 12 days the newly differentiated CD31⁺ cells,which had been cultivated on Matrigel®, form a vessel-likethree-dimensional tube with multi-layer wall structures, which isalready morphologically reminiscent of a vessel. It is recognised, thatnow almost all the cells express the CD31 antigen (CD31 coloration,×400, blue filter), right-hand picture.

EXAMPLE 6 Production of Fat Cells (Adipocytes)

[0184] A: For the programming/differentiation of the adult stem cellsaccording to Example 2 into fat cells, a conditioned medium was firstgenerated. For this purpose 20 g of an autologous fat tissue, i.e. fattissue from the same human donor, from the blood of whom the monocytesalso originated, was processed as follows:

[0185] At first, the fat tissue was crushed in a petri dish and thecrushed tissue pieces were passed through a sieve (diameter of holes 100μm).

[0186] The suspension thus obtained was then transferred into a petridish with a diameter of 100 mm and 10 ml DMEM-medium with a content of30 mg collagenase type II were added. The mixture was left forapproximately 60 minutes at room temperature (22° C.±2° C.) to allow thecollagenase to take effect on the fat cells.

[0187] Subsequently the mixture was transferred to 50-ml Falcon tubes,and the tubes were centrifuged for 10 minutes at 1800 rpm.

[0188] After centrifugation the supernatant was discarded and the cellpellet consisting of adipocytes and precursor cells was taken up in 8 mlof a medium of the following composition and incubated in petri dishes(diameter 100 mm) for 10 days at 37° C. in an incubator: DMEM solution444.5 ml Foetal calf serum (FCS)   50 ml Insulin solution  0.5 mlPenicillin (100 U/l)/Streptomycin    5 ml (100 μg/l) solution Totalvolume   500 ml

[0189] The insulin solution contained 18 mg insulin (Sigma 1-0259)dissolved in 2 ml of acetic water (consisting of 40 ml of H₂O and 0.4 mlof glacial acetic acid). The solution is diluted 1:10 with acetic water.

[0190] During the incubation over 10 days, the fat-cell-conditionedmedium (FCCM) formed a supernatant. The supernatant was replaced withfresh nutrient medium after 2 to 4 days in each case. The FCCM obtainedduring each change of medium was subjected to sterile filtration andstored at −20° C. Subsequently 10 ml of the FCCM described above wereintroduced into a petri dish (diameter 100 mm) together withapproximately 10⁶ stem cells according to Example 2. The first precursorcells containing fat vacuoles became visible after 4 days (FIG. 3A).After 6 days, single adipocytes appeared, which could be stained withSudan red (FIGS. 3B and C). After 10 days there was typical aggregationand cluster formation of these cells, which at this step could alreadybe observed macroscopically as fat tissue (FIG. 3D).

[0191] The fat cells made visible by staining in FIGS. 3A-3D thus differquite considerably from the controls 3E and 3F: FIG. 3E shows the cellsof monocytic origin, which were cultivated in the nutrient medium (asindicated in Example 2) for 6 days, but without the addition of IL-3 and2-mercaptoethanol to the nutrient medium. This was followed by theaddition of the FCCM. These cells were not capable of differentiatinginto fat cells. Figure F. shows cells, which were cultivated for 6 dayswith complete medium (according to Example 2), and which were thentreated for a further 6 days with nutrient medium instead of with FCCM(according to Example 2). The FCCM thus contains components which arerequired to provide the signal for differentiation into fat cells.

[0192] The staining of the cells with Sudan red in FIGS. 3A, B, C and Dtook place according to the method described by Patrick Jr., C. W., etal. “Epithelial Cell Culture: Breast”, in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 141-149.

[0193] B: In addition to the phenotyping of the fat cells by stainingwith Sudan red, molecular-biological characterisation of the fat cellswas carried out at the mRNA level, in order to check whether the geneticprogramme of the fat cells, after corresponding programming with thefat-cell-conditioning medium used, undergoes a corresponding alteration,and typical messenger-ribonucleic acid (mRNA) transcripts, described forfat cells can be identified in the fat cells programmed fromprogrammable monocytes. Two mRNA sequences typical of fat cellmetabolism were amplified by means of polymerase chain reaction (PCR)from isolated RNA samples from dedifferentiated programmable stem cellsof monocytic origin and, in a parallel test mixture, amplified from theprogrammed fat cells, namely “peroxisome proliferative activatedreceptor gamma” (PPARG)-mRNA, (Tontonoz, P., et al. “Stimulation ofadipogenesis in fibroblasts by PPAR gamma 2, a lipid-activatedtranscription factor.” Cell 79: 1147-1156 (1994), gene bank access codenumber; NM_(—)005037) and “leptin (obesity homolog, mouse)”-mRNA, (ZhangY., et al. “Positional cloning of the mouse obese gene and its humanhomologue.” Nature 372: 425-432 (1994), gene bank, access code number:NM_(—)000320).

[0194] The RNA-isolation needed for this purpose, the reversetranscription method and the conditions of the PCR amplification of thedesired mRNA sequences were carried out as described in detail in thestate of the art, see Ungefroren H., et al., “Human pancreaticadenocarcinomas express Fas and Fas ligand yet are resistant toFasmediated apoptosis”, Cancer Res. 58: 1741-1749 (1998).

[0195] For this purpose the respective primers produced for the PCRamplification were selected so that the forward- and reverse primersbind to mRNA sequences, whose homologous regions in the chromosomal genelie in two different exons and are separated from one another by a largeintron. It could thereby be ensured that the amplification fragmentobtained originates from the mRNA contained in the cell and not from thesequence present in the chromosomal DNA. In particular the followingprimer sequences were selected for PPAR-γ and for leptin:

[0196] PPAR-γ: forward-primer; 265-288 (corresponding gene sequence inexon 1), reverse-primer: 487-465 (corresponding gene sequence in exon2), this results in an amplification fragment of 487-265 bp=223 bp, seeFIG. 3G. As further shown by FIG. 3G traces of transcribedPPAR-γ-specific mRNA can already be identified in the programmable stemcell and in the tumor cell line HL-60 (of a human promyeloic leukaemiacell line), although with significantly narrower signal bands than inthe fat cell itself. In contrast, the fat-cell-specific protein leptincan only be detected in the fat cells derived from the programmable stemcells at mRNA level by reverse-transcriptase PCR.

[0197] The programmable stem cells (progr. stem cell) used as a controland the human tumour cell lines HL-60, Panc-1 and WI-38 transcribe noleptin. As negative controls all the samples without the addition of thereverse transcriptase (fat cell/−RT) and H₂O-samples were simultaneouslycodetermined. By identification of the GAPDH “house-keeping” gene in thepositive controls, it is ensured that the respective PCR amplificationsteps were properly carried out in the individual mixtures.

EXAMPLE 7 Production of Liver Cells (Hepatocytes)

[0198] A: For the programming of the dedifferentiated programmable stemcells of monocytic origin according to Example 2 into liver cells, aconditioned medium was first generated. For this purpose 40 g of humanliver tissue was processed as follows.

[0199] First the liver tissue was rinsed several times in PBS, toessentially remove erythrocytes. The tissue was then crushed in a petridish and incubated with a dissociation solution for approximately 45minutes at room temperature. The dissociation solution consisted of 40ml PBS (phosphate buffered saline), 10 ml of a trypsin solution diluted1:10 with PBS and 30 mg collagenase type II (Rodbel M., et al. J. Biol.Chem. 239: 375 (1964)). After 45-minutes' incubation the tissue pieceswere passed through a sieve (see Example 6).

[0200] The mixture was then transferred into 50-ml Falcon tubes, filledup to 50 ml with PBS and centrifuged for 10 minutes at 1800 rpm.

[0201] After centrifugation the supernatant was discarded and the cellpellet containing the liver cells was again washed with 50 ml PBS andcentrifuged. The supernatant thus produced was again discarded and thecell pellet taken up in 25 ml of a medium of the following compositionand incubated in cell culture flasks (250 ml volume) for 10 days at 37°C. in an incubator:

[0202] Liver Cell Growth Medium Liver cell growth medium, LCGM RPMI 1640medium 445 ml Foetal calf serum (FCS)   50 ml Insulin solution  0.5 mlPenicillin(100 U/l)/Streptomycin (100 μg/l) solution   5 ml Total volume 500 ml

[0203] The nutrient medium contained in addition 5 μg (10 ng/ml) ofepidermal growth factor (Pascall, I. C. et al., J. Mol. Endocrinol. 12:313 (1994)). The composition of the Insulin solution was as described inExample 6.

[0204] During the incubation lasting 10 days the liver cell conditionedmedium (LCCM) formed as a supernatant. The supernatant was replaced byfresh nutrient medium after 2 to 4 days respectively. The respectiveLCCM obtained during the change of medium in each case was subjected tosterile filtration (filter with 0.2 μm pore size) and stored at −20° C.

[0205] 1×10⁶ dedifferentiated stem cells were then cultivated with 10 mlof a medium of the following composition in a petri dish (ø100 mm) or aculture flask.

[0206] Liver Cell Differentiation Medium (Liver cell differentiationmedium, LCDM): LCCM 100 ml Insulin solution (cf. Example 6) 0.1 mlepidermal growth factor 1 μg hepatocyte growth factor 2 μg

[0207] Hepatocyte growth factor (Kobayashi, Y. et al., Biochem. Biophys.Res. Commun. 220: 7 (1996)) was used in the concentration of 40 ng/ml.After a few days morphological changes towards flat, polygonal mono- ordiploid cells could be observed (FIG. 4A). After 10-12 days hepatocytesarising from dedifferentiated stem cells could be identified byimmune-histochemical detection of the liver-specific antigenalpha-fetoprotein (Jacobsen, G. K. et al., Am. J. Surg. Pathol. 5:257-66 (1981)), as shown in FIGS. 4B and 4C.

[0208] B: In addition to the phenotyping of the hepatocytes byimmune-histochemical identification of the alpha-fetoprotein, amolecular-biological characterisation of the hepatocytes at mRNA levelwas carried out, in order to check whether the genetic programme of thestem cells, after corresponding programming with theliver-cell-conditioning medium used undergoes a correspondingalteration, and whether messenger-ribonucleic acid (mRNA) transcripts,described as typical of liver cells in the hepatocytes arising from thestem cells according to the invention can be identified. For thispurpose, the presence of five different mRNA sequences typical ofhepatocytes was examined by means of polymerase chain reaction (PCR) inisolated RNA samples from dedifferentiated programmable stem cells ofmonocytic origin and, in a parallel test sample, from the liver cellsobtained by programming of the stem cells. In particular, this is theHomo sapiens albumin-mRNA (Lawn, R. M., et al. “The sequence of humanserum albumin cDNA and its expression in E. coli.” Nucleic Acids Res. 9:6103-6114, (1981), gene bank access code number: NM-000477),alpha-fetoprotein-mRNA (Morinaga T., et al. “Primary structures of humanalpha-fetoprotein and its mRNA.” Proc. Natl. Acad. Sci. USA 80:4604-4608 (1983), gene bank access code number: V01514), Human carbamylphosphate synthetase I mRNA (Haraguchi, Y., et al. “Cloning and sequenceof a cDNA encoding human carbamyl phosphate synthetase I: molecularanalysis of hyperammonemia” Gene 107: 335-340 (1991), gene bank accesscode number D90282), Homo sapiens coagulation factor II (Thrombin, F2)mRNA (Degen, S. J. et al. “Characterization of the complementarydeoxyribonucleic acid and gene coding for human prothrombin”Biochemistry 22: 2087-2097 (1983), gene bank access code numberNM-000506), Homo sapiens coagulation factor VII (serum prothrombinconversion accelerator, F7) mRNA (NCBI Annotation Project. DirectSubmission, 06-Feb.-2002, National Center for Biotechnology Information,NIH, Bethesda, Md. 20894, USA, gene bank access code number XM-027508).

[0209] The RNA-isolation necessary for this reverse transcriptase methodand the conditions of the PCR amplification of the desired mRNAsequences was carried out as described in detail in the state of theart, see Ungefroren H., et al., “Human pancreatic adenocarcinomasexpress Fas and Fas ligand yet are resistant to Fas-mediated apoptosis”Cancer Res. 58: 1741-1749 (1998).

[0210] The respective primers for the PCR amplification were selected sothat the forward- and reverse primers bind to mRNA sequences whosehomologous regions in the chromosomal gene lie in two different exonsand are separated from one another by a large intron. In this way itcould be ensured that the amplification fragment obtained originatesfrom the mRNA contained in the cell and not from the sequence present inthe chromosomal DNA.

[0211] The primer sequences indicated below were selected; the resultsof the respective PCR analyses are reproduced in FIG. 4D. Thededifferentiated programmable stem cells according to the invention, aredesignated there as “progr. stem cell” and the hepatocytes derived byprogramming of these as “progr. hepatocyte”.

[0212] Alpha-fetoprotein: forward primer: 1458-1478 (corresponding genesequence in Exon 1), reverse primer: 1758-1735 (corresponding genesequence in Exon 2), this results in an amplification fragment of1758-1458 bp=391 bp, see FIG. 4D.

[0213] As shown in FIG. 4, the programmable stem cell (progr. stemcell), which itself contains no identifiable specific mRNA transcriptsfor alpha-fetoprotein, can be programmed into a hepatocyte (progr.hepatocyte), which contains this mRNA transcript (positive band with amolecular weight of 301 bp). This also explains the immune-histochemicaldetectability of the alpha-fetoprotein, as shown in FIGS. 4B and 4C. Thepositive controls, namely human liver tissue and the liver tumour cellline HepG2 also transcribe alpha-fetoprotein-specific mRNA, as the 301bp bands confirm.

[0214] Albumin: forward primer: 1450-1473 (corresponding gene sequencein exon 1), reverse primer: 1868-1844 (corresponding gene sequence inExon 2), this resulted in an amplification fragment of 1868-1450 bp=419bp, see FIG. 4D.

[0215]FIG. 4D shows traces of transcribed albumin-specific mRNA alreadyin the programmable stem cell, whilst the hepatocytes obtained byprogramming of the stem cells and normal liver tissue as well as thetumour cell line HepG2, which were both used as positive controls,strongly express the mRNA, as can be recognized by clear bands.

[0216] The carbamyl phosphatase synthetase I: forward primer: 3135-3157(corresponding gene sequence in exon 1), reverse primer: 4635-4613(corresponding gene sequence in exon 2), this results in anamplification fragment of 4635-3135=1500 bp, see FIG. 4D.

[0217] The carbamyl phosphate synthetase I represents an enzyme specificto the hepatocytes, which plays an important role in the metabolisationof urea in the “urea cycle”. This detoxification function is guaranteedby functioning hepatocytes. As FIG. 4D shows, both in the hepatocytesgenerated from programmable stem cells and also in the positive controls(human liver tissue and the HepG2-tumour cell line), the mRNA bands(1500 bp) specific to carbamyl phosphate synthetase I can be identified.The somewhat weaker expression of the mRNA bands for the programmedhepatocytes (progr. hepatocyte) is due to the lack of substrateavailable in the culture dish.

[0218] Clotting factor II: forward primer: 1458-1481 (corresponding genesequence in exon 1), reverse primer: 1901-1877 (corresponding genesequence in exon 2), this results in an amplification fragment of1901-1458=444 bp, see FIG. 4D.

[0219] This likewise hepatocyte-specific protein can only be detected inthe programmed hepatocyte (progr. hepatocyte) and in the positivecontrol from human liver tissue at mRNA level by 444 bp band expression,whereas the programmable stem cell (progr. stem cell) does not show thisband, i.e. the gene is not transcribed there, as can be seen in FIG. 4D.

[0220] Clotting factor VII: forward primer: 725-747 (corresponding genesequence in exon 1), reverse primer: 1289-1268 (corresponding genesequence in exon 2), this results in an amplification fragment of1289-725=565 bp, see FIG. 4D.

[0221] As in the case of clotting factor II, also this protein is onlytranscribed in programmed hepatocytes (progr. hepatocyte) and in thepositive control (human liver tissue) (see bands at 656 bp), althoughweaker than clotting factor II. Neither the programmable stem cell northe negative control (H₂O) show this specific mRNA band.

[0222] Glycerine aldehyde dehydrogenase: This gene, also referred to asa “house-keeping gene” can be detected in every eukaryotic cell andserves as a control whether PCR amplification was properly carried outin all samples; it is co-determined in parallel and results from theaddition of a definite quantity of RNA from the respective cell samples.

[0223] As negative control H₂O samples were simultaneously codeterminedin all tests. If the H₂O is not contaminated with RNA, no amplificate isproduced during the PCR and no band is detectable (thus serves ascounter-control).

EXAMPLE 8 Production of Skin Cells (Keratinocytes)

[0224] For the programming of dedifferentiated programmable stem cellsof monocytic origin according to Example 2 in skin cells a conditionedmedium was first generated. For this purpose, 1-2 cm² of complete humanskin was processed as follows.

[0225] The skin material was first freed from the subcutis under sterileconditions. The tissue was then washed in total 10× with PBS in asterile container by vigorous shaking. After the 2nd washing, the tissuewas again freed from demarked connective tissue residues.

[0226] The skin material was then placed in a petri dish with a diameterof 60 mm, mixed with 3 ml of a trypsin solution diluted 1:10 with PBSand cut into small pieces (approximately 0.5 to 1 mm³). After this, 3 mlof the trypsin solution diluted 1:100 with PBS was again added to themixture and the mixture was incubated at 37° C. for 60 minutes withintermittent shaking.

[0227] The larger particles were then allowed to settle and thesupernatant containing the keratinocytes was poured off and centrifugedat 800 rpm for 5 minutes. The supernatant now produced was pipetted offand the cell pellet was taken up in 3 ml of a medium of the followingcomposition and incubated in petri dishes (ø100 mm) for 15 days in anincubator at 37° C.

[0228] Keratinocyte Growth Medium (Keratinocyte growth medium, KGM):DMEM  333 ml Foetal Calf serum (FCS)   59 ml Ham's F12 medium  111 mlPenicillin (100 U/l)/Streptomycin (100 μg/l) solution   5 ml Insulinsolution (cf. Example 6)  0.5 ml Total volume  500 ml

[0229] The nutrient medium contained 5 μg of epidermal growth factor(for exact specification see Example 7) and 5 mg of hydrocortisone (Ref.Merck Index: 12, 4828).

[0230] During the 15 days' incubation period, thekeratinocyte-cell-conditioned medium KCCM formed as supernatant. Thesupernatant was replaced with fresh nutrient medium after 2-4 days ineach case. The KCCM obtained during each change of medium was subjectedto sterile filtration and stored at −20° C.

[0231] 1×10⁶ dedifferentiated stem cells were then cultivated with 10 mlof a mediums of the following composition in a petri dish (ø100 mm) or aculture flask.

[0232] Keratinocyte Differentiation Medium Keratinocyte differentiationmedium (Keratinocyte differentiation medium, KDM) KCCM 100 ml Insulinsolution (cf. Example 6) 0.5 ml epidermal growth factor (EGF) 1 μgHydrocortisone 1 mg keratinocyte growth factor (KGF) 2.5 μg

[0233] Keratinocyte growth factor was used in a concentration of 25ng/ml, as described by Finch et al., Gastroenterology 110: 441 (1996).

[0234] After a few days a morphological change in the cells could beobserved. After 6 days the keratinocyte-specific antigens, cytokeratin 5and 6, which are both bound by the primary antibody used, (Exp. Cell.Res. 162: 114 (1986)) could be detected (FIG. 5A). After 10 days a celladherence of the clearly larger individual cells already took place inculture, which made it possible to identify a visible cell tissuecombination of confluent cells (FIG. 5B).

EXAMPLE 9 Production of Insulin-Producing Cells from DifferentiatedProgrammed Stem Cells

[0235] The production of insulin-producing cells was conducted inculture flasks with a volume of approximately 250 ml and flat walls (T75cell culture flasks). Approximately 5×10⁶ of the cells producedaccording to Example 13 were suspended in approximately 5 ml of theculture medium indicated below (differentiation medium for insulinproducing cells) and after being introduced into the flasks, mixed witha further 15 ml of culture medium. For the differentiation of the cells,the flasks were incubated in a horizontal position in an incubator at37° C. and 5% CO₂.

[0236] Culture medium (modified according to Rameya V. K. et al., NatureMedicine, 6 (3), 278-282 (2000)): RPMI 1640 445 ml Foetal calf serum(FCS) 50 ml Penicillin (100 U/l)/Streptomycin 5 ml (100μg/l) solutionNicotinamide 620 mg Glucose 360 mg Total volume 500 ml

[0237] The nutrient medium further contained the epidermal growth factorin a quantity of 10 ng/ml and the hepatocyte growth factor in a quantityof 20 ng/ml.

[0238] Within the first hour the cells adhere to the bottom of theculture vessel. The differentiation of the stem cells was monitored byreference to insulin production. For this purpose the culture medium waschanged at intervals of approximately 2 to 3 days, the cell supernatantwas collected each time, and frozen at −20° C. The cells adhering to thebottom of the culture flask could be detached by tryptinisation asdescribed in Example 2.

[0239] The insulin content of the supernatant collected at the differenttimes was measured by means of ELISA (Enzyme-linked-immunosorbent-assay)against human insulin (Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch derLabormedizin: Grundlagen, Diagnostik, Klinik Pathobiochemie [Textbook ofLaboratory Medicine, Principles, Diagnosis, Clinical Pathobiochemistry](1999), Page 189) and compared with the blank reading of the medium. Theresults reproduced in FIG. 8 show that the cells have reached themaximum level of insulin production after 14 days in culture. Theinsulin quantities produced by the cells treated in the course of thedifferentiation increased after 14 days to 3 μU/ml, whilst no humaninsulin was detectable in the control medium. The bars in FIG. 8 eachrepresent three separate values each determined from three independentindividual experiments.

[0240] Next to the determination of the insulin production in thedeprogrammed stem cells, which were differentiated into insulinproducing cells according to the invention, the portion ofinsulin-producing cells were determined which still expressed themonocyte-specific surface antigen CD14 also 3 weeks after conducting thededifferentiation. It was found that on a great portion of these cells(about 30 to 40%) the monocyte-specific antigen CD14 was detectable alsoafter 3 weeks.

EXAMPLE 10 Alternative Method for the Production of Hepatocytes fromDedifferentiated Programmable Stem Cells

[0241] As an alternative to the use of hepatocyte-conditioned medium(LCCM), as described in Example 7, the differentiation of the stem cellsinto hepatocytes was induced by the nutrient medium (Ha) indicatedbelow. The production of hepatocytes from stem cells in turn took placein culture flasks with a volume of approximately 250 ml and flat walls(T75-cell culture flasks). Approximately 5×10⁶ of the cells producedaccording to Example 13 were introduced into approximately 5 ml of theimproved culture medium indicated below (Ha, differentiation medium forhepatocytes) and after being introduced into the flasks, mixed with afurther 15 ml of culture medium. For the differentiation of the cells,the flasks were incubated in a horizontal position in an incubator at37° C. and 5% CO₂.

[0242] Differentiation medium for hepatocytes (Ha) (modified accordingto Schwarz et al., “Multipotent adult progenitor cells from bone marrowdifferentiate into functional hepatocyte-like cells”, J. Clin. Invest.10 (109), 1291-1302 (2002)): RPMI 1640 445 ml Foetal calf serum (FCS) 50 ml Penicillin (100 U/l)/  5 ml Streptomycin (100 μg/l) solutionTotal volume 500 ml

[0243] The nutrient medium also contained fibroblast growth factor-4(FGF-4) in a quantity of 3 ng/ml.

[0244] Within the first hour the cells adhere to the bottom of theculture vessel. The differentiation of the stem cells was monitored withregard to albumin production. For this purpose the culture medium waschanged at intervals of approximately 2 to 3 days, the cell supernatantcollected each time, and frozen at −20° C. The cells adhering to thebase of the culture flask could be detached by tryptinisation asdescribed in Example 2.

[0245] The albumin content of the supernatant collected at the differenttimes was measured by means of ELISA (Enzyme-linked-immunosorbent-assay)for human albumin (according to the protocol of Bethyl Laboratories Inc.and according to Schwarz et al., loc. cit.) and compared with the blankreading of the medium. The results presented in FIG. 9 show that thealbumin production of the cells during the period of 14 to 28 days inculture remained approximately constant. The measurements were carriedout on days 0 (blank reading of the medium), 14, 21, 28 and 30 relativeto the time of addition of the Ha medium. The values determined in eachcase amounted to ca. 5 ng/ml, 450 ng/ml, 425 ng/ml, 440 ng/ml and 165ng/ml. The bars in FIG. 9 each represent three separate values eachdetermined from three independent individual experiments.

EXAMPLE 11 Detetermination of the Co-Expression of Albumin and of theMonocyte-Specific Antigen CD14 in Hepatocytes Derived fromDedifferentiated Stem Cells

[0246] The determination of the co-expression of albumin and of themonocyte-specific antigen CD14 in hepatocytes derived fromdedifferentiated stem cells was carried out on the one hand bydouble-staining (A) and on the other hand by FACS analysis (B).

[0247] A) Stem cells according to the invention differentiated intohepatocytes according to Example 10 were cultivated on cover glasses ina 6-well plate and fixed with methanol as described in Example 4. Adouble-staining was then carried out, in order to detect thesimultaneous expression of the antigen CD14 (phenotype marker ofmonocytes) on the one hand and of albumin (liver-specific marker) on theother hand.

[0248] For this purpose the cells were first incubated as described inExample 4 with a primary antibody against human albumin (guinea pig vs.human albumin) in a 1:50 dilution in PBS. Following a washing step, thecells were then incubated for 45 minutes with a secondary antibody mouseanti-rat, which binds the guinea pig antibodies, also in a 1:50 dilutionin PBS. The staining process was then carried out according to Example 4using the method of Cordell J. L., et al. (loc. cit.) with APAAP redcomplex.

[0249] For the second staining step, the cells were then incubated withthe primary antibody, mouse anti-human-CD14, and following a washingstep according to Example 4 stained with the ABC Streptavidin KIT ofVectastain (Vector) using the method of Hsu, S. M., et al. “The use ofantiavidin antibody and avidin-biotin-peroxidase complex inimmunoperoxidase technics” Am. J. Clin. Pathol. 75 (6): 816-82.1 (1981)with dem DAB-Complex (brown) (Vector Laboratories).

[0250] Nucleus counter-staining with haemalaun was then carried out asdescribed in Example 4, followed by embedding in Kaiser's glycerolgelatin.

[0251] The results are shown in FIG. 10. The figure shows the expressionof the antigen CD14 as brown color, which slowly decreases parallel tothe morphological transformation of the cells into hepatocytes, whilstthe albumin expression as red color increases with the increasingmaturation of the hepatocytes. Picture No.4 in FIG. 10 shows the cellsafter three weeks' stimulation with the hepatocyte-conditioned medium.

[0252] B) Parallel with the double marking, the stem cellsdifferentiated into hepatocytes according to the invention weresubjected to FACS (fluorescence-activated cell sorting) analysis.

[0253] The stem cells differentiated into hepatocytes according to theinvention according to Example 10 were first harvested by mechanicaldetachment of the cells from the culture flask using a cell scraper. Thecells were carefully rinsed from the flask with PBS and washed twice,each time in 10 ml of PBS-solution. For this purpose the cellsuspensions in the PBS solution were introduced into a 15-ml centrifugetube and precipitated at 1600 rpm. The resultant cell sediment wasdiluted with PBS, such that exactly 1×10⁵ cells were present in 100 μlPBS.

[0254] 10 μl of each of FITC-marked anti-CD14 antibodies (BD Pharmingen)or FITC-marked anti-albumin antibodies (Beckmann) and FITC-markednon-specific IgG1 mouse antihuman antibodies were then added to thiscell suspension. After an incubation period of 20 minutes the cells wereresuspended twice in 500 μl PBS and each precipitated for 5 minutes at1600 rpm and then finally taken up in 200 μl PBS. After resuspension ofthe cells, fluorescence was measured with a BD FACScalibur flowcytometer from the company BD Biosciences (Franklin Lakes, N.J.) (cf.Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch der Labormedizin: Grundlagen,Diagnostik, Klinik Pathobiochemie [Textbook of Laboratory Medicine,Principles, Diagnosis, Clinical Pathobiochemistry], 395-403 (1999); andHolzer U. et al., “Differential antigen sensitivity and costimulatoryrequirements in human Th1 and Th2 antigen-specific CD4(+) cells withsimilar TCR avidity” J. Immunol. 170 (3): 1218-1223 (2003)). Theevaluation of the results was carried out using the Microsoft WinMDIprogram with reference to Marquez M. G., et al. “Flow cytometricanalysis of intestinal intra-epithelial lymphocytes in a model ofimmunodeficiency in Wistar rats.” Cytometry 41 (2): 115-122 (2000).

[0255] The results of the FACS-Analyse are reproduced in FIG. 11. Thefigure shows the expression of the CD14 (top row) and of the albuminantigen (bottom row), which was measured in dedifferentiated monocytes(left-hand column) and in the stem cells differentiated into hepatocytesaccording to the invention (right-hand column). In dedifferentiatedmonocytes a strong expression of CD14, but no expression of albumincould be detected, whilst in the hepatocytes developed fromdedifferentiated monocytes a weaker expression of the CD14 and a verystrong expression of the albumin was detectable.

EXAMPLE 12 In Vivo Use of Dedifferentiated Programmed Stem Cells ofMonocytic Origin

[0256] In order to clarify, to what extent the programmable stem cellsin vivo after injection via the portal vein into the liver of agenetically identical recipient animal undergo a specificdifferentiation via the signal-providers present in the liver, livers offemale LEW rats were first treated with retrorsine, in order to inhibitthe hepatocytes present in the liver (liver parenchyma cells) regardingtheir proliferation activity (Ref. Lacone, E., et al. “Long-term,near-total liver replacement by transplantation of isolated hepatocytesin rats treated with retrorsine” Am. J. Path. 153: 319-329 (1998)).

[0257] For this purpose the LEW rats received 30 mg of the pyrrolizidinealkaloid retrorsine, injected intraperitoneally twice within 14 days.Subsequently an 80% resection of the livers treated in this way wascarried out, followed by the administration of 5×10⁵ of the programmablestem cells in 1 ml PBS into the portal vein of the remaining residualliver. The stem cells had been obtained, as described in Example 2, frommonocytes of male LEW rats. Five days after administration of the stemcells a punch biopsy of the liver was carried out for histologicalassessment of the liver and to detect the cell types differentiated fromthe stem cells by means of fluorescence-in-situ-hybridisation (FISH)with Y-chromosome-specific probes, as described in detail in Hoebee, B.et al. “Isolation of rat chromosome-specific paint probes by bivariateflow sorting followed by degenerate oligonucleotide primed-PCR.”Cytogenet. Cell Genet. 66: 277-282 (1994).

[0258]FIG. 7A shows the Y-chromosome-positive (red points in the cellnucleus) hepatocytes derived from the male LEW stem cells on the 5th dayafter intraportal injection into retrorsinepretreated 80%-resectionedlivers of female recipient animals. The selective removal of the sameliver on day 25 after stem cell injection shows the differentiation ofthe stem cells into hepatocytes, endothelial cells and bile ductepithelia. At this point in time, the liver has already reached itsnormal size, and >90% of the cells have a Y-chromosome. From this, itcan be concluded, that the injected syngenic programmable stem cells ofmonocytic origin in are capable in vivo, of effecting a completerestoration of the liver with normal metabolic function. FIG. 7C showsin this connection the Kaplan-Meier survival curves (n=4 per group) ofstem-cell-treated versus untreated recipient rats followingadministration of retrorsine and 80% liver resection.

[0259] The function parameters bilirubin and ammonia (NH₃) prove thecomplete metabolic functionality of the long-term survivingstem-cell-treated animals (FIGS. 7D and 7E).

EXAMPLE 13 Propagation and Dedifferentiation of Monocytes in CellCulture Flasks

[0260] Cultivation and propagation of the monocytes on the one hand andthe dedifferentiation of the cells of the other side on a larger scalewere conducted in culture flasks in the same nutrient medium, which wasalso used for the cultivation in well-plates (cf. Example 2). Thenutrient medium containt 2.5 μg/500 ml M-CSF and 0.2 μg/500 mlinterleukine 3 (IL-3).

[0261] The monocytes isolated in Example 1 were transferred to thebottom of culture flasks having a volume of 250 ml and flat walls(T75-cell culture flasks). About 10 times×10⁶ cells were transferredinto each flasks and were each filled up with 20 ml of the aboveindicated nutrient medium. The determination of this cell number for theexact dosing per flask was carried out according to known procedures,cf. Hay R. J., “Cell Quantification and Characterization” in Methods ofTissue Engineering, Academic Press (2002), Chapter 4, pages 55-84.

[0262] The cell culture flasks were incubated in an incubator at 37° C.for 6 days. After 24 hours, the cells settled at the bottom of theflasks. The supernatant was removed every second day and the flasks wereeach filled with 20 ml fresh nutrient medium.

[0263] On day 6, the flasks were rinsed twice with 10 ml PBS each, afterthe nutrient medium had previously been pipetted off from the flasks.Hereby, all cells were removed, which did not adhere to the bottom ofthe flasks. The cells growing adhere to the bottom of the flasks weresubsequently removed from the bottom of the flasks with a steril cellscraper. The separated cells were now removed from the flasks by rinsingwith PBS and were pooled in a 50 ml Falcon tube and were centrifuged at1800 rpm for 10 minutes. Thereafter, the supernatant was discarded andthe sediment was resuspended in fresh RPMI 1640 medium (2 ml/10⁵ cells).

[0264] This cell suspension could be used directly for differentiatinginto various target cells.

[0265] Alternatively, the cells were mixed with DMSO/FCS as freezingmedium after centrifugation and were deep-frozen at a concentration of10⁶/ml.

[0266] The freezing medium contained 95% FCS and 5% DMSO. About 10⁶cells were taken up in 1 ml of the medium and were cooled following thesubsequent steps:

[0267] 30 minutes on ice;

[0268] 2 hours at −20° C. in precooled styropor box;

[0269] 24 hours at −80° C. in styropor;

[0270] stored in tubes in liquid nitrogen (N₂) at −180° C.

1. Process for the production of dedifferentiated, programmable stemcells of human monocytic origin, characterised in that a) monocytes areisolated from human blood; b) the monocytes are propagated in a suitableculture medium, which contains the cellular growth factor M-CSF; c) themonocytes are cultivated simultaneously with or subsequently to step b)in a culture medium containing IL-3; and d) the human adultdedifferentiated programmable stem cells are obtained by separating thecells from culture medium.
 2. Process according to claim 1,characterised in that a mercapto compound is further added to theculture medium in step c).
 3. Process according to claim 2,characterised in that a mercapto compound is used, in which at least onecarbon group is bonded to the sulphur, and wherein the hydrocarbongroup(s) may be substituted with one or more further funtional groups.4. Process according to claims 2 or 3, characterised in that themercapto compound is 2-mercaptoethanol or Dimethylsulfoxide.
 5. Processaccording to claims 1 to 4, characterised in that subsequent to step c)and before step d) the cells are contacted with a biologicallyacceptable organic solvent.
 6. Process according to claim 5,characterised in that the biologically acceptable organic solvent is analcohol with 1-4 carbon atoms.
 7. Process according to claim 6,characterised in that the alcohol is ethanol.
 8. Process according toclaims 5 to 7, characterised in that the cells are brought into contactwith the vapour phase of the biologically acceptable organic solvent. 9.Process according to claims 1 to 8, characterised in that the cells aresuspended in a suitable cell culture medium subsequent to step d). 10.Processaccording to claim 9, characterised in that the medium is RPMI orDMEM.
 11. Process according to claims 9 or 10, characterised in that themedium contains a cytokine or LIF.
 12. Process according to claims 9 to11, characterised in that the cells are suspended in a liquid medium andsubsequently deep frozen.
 13. Process according to claim 12,characterised in that the medium is a cell culture medium. 14.Dedifferentiated, programmable stem cells of human monocytic origin. 15.Stem cells according to claim 14, obtainable by the process of claims 1to
 13. 16. Pharmaceutical composition, containing the dedifferentiated,programmable stem cells according to claims 14 or
 15. 17. Use of thededifferentiated, programmable stem cells according to claims 14 or 15for producing target cells and target tissue.
 18. Use according to claim17, characterised in that a) the tissue containing the desired targetcells is crushed; b) the target cells and/or fragments thereof areobtained from the crushed tissue; c) the target cells and/or fragmentsthereof are incubated in a suitable culture medium; d) the supernatantof the culture medium is collected during and after incubation astarget-cell-conditioned medium; and e) for thereprogramming/differentiation of the stem cells into the desired targetcells, the stem cells are allowed to grow in the presence of thetarget-cell-conditioned medium.
 19. Use according to claims 17 or 18,for the production of adiocytes, of neurons and glia cells, ofendothelial cells, of keratinocytes, of hepatocytes or of islet cells.20. Process according to claims 1 to 13, characterised in that thededifferentiated, programmable stem cells are transfected with one ormore genes.
 21. Dedifferentiated, programmable stem cells of humanmonocytic origin according to claim 14, characterised by the membraneassociated monocyte-specific surface antigen CD 14 and at least onepluripotency marker selected from the group consisting of CD90, CD117,CD123 and CD135.
 22. Stem cells according to claims 14, 15 or 21,characterised in that the dedifferentiated, programmable stem cells aretransfected with one or more genes.
 23. Stem cell preparation,containing dedifferentiated, programmable stem cells according to claims14, 15, 21 or 22 in a suitable medium.
 24. Use of the dedifferentiated,programmable stem cells according to claims 14, 15, 21 or 22 for thepreparation of a pharmaceutical composition for the treatment of livercirrhosis.
 25. Use of the dedifferentiated, programmable stem cellsaccording to claims 14, 15, 21 or 22 for the preparation of apharmaceutical composition for the treatment of pancreaticinsufficiency.
 26. Use of the dedifferentiated, programmable stem cellsaccording to claims 14, 15, 21 or 22 for the preparation of apharmaceutical composition for the treatment of acute or chronic kidneyfailure.
 27. Use of the dedifferentiated, programmable stem cellsaccording to claims 14, 15, 21 or 22 for the preparation of apharmaceutical composition for the treatment of hormonalunder-functioning.
 28. Use of the dedifferentiated, programmable stemcells according to claims 14, 15, 21 or 22 for the preparation of apharmaceutical composition for the treatment of cardiac infarction. 29.Use of the dedifferentiated, programmable stem cells according to claims14, 15, 21 or 22 for the preparation of a pharmaceutical composition forthe treatment of pulmonary embolisms.
 30. Use of the dedifferentiated,programmable stem cells according to claims 14, 15, 21 or 22 for thepreparation of a pharmaceutical composition for the treatment of stroke.31. Use of the dedifferentiated, programmable stem cells according toclaims 14, 15, 21 or 22 for the preparation of a pharmaceuticalcomposition for the treatment of skin damage.
 32. Use of thededifferentiated, programmable stem cells according to claims 14, 15, 21or 22 for the preparation of a pharmaceutical composition for the invivo production of target cells and target tissue.
 33. Differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to claims 14, 15, 21 or 22,characterised by the membrane-associated surface antigen CD14. 34.Somatic target cells and/or target tissue according to claim 33,selected from the group consisting of adipocytes, neurons and gliacells, endothelial cells, keratinocytes, hepatocytes and islet cells.35. Somatic target cells and/or target tissue according to claims 33 or34, characterised in that they are transfected with one or more genes.36. Implantable materials coated with the dedifferentiated, programmablestem cells according to claims 14, 15, 21 or 22 or the somatic targetcells and/or target tissue according to 33 to
 35. 37. Implantedmaterials according to claim 36, characterised in that the materials areprostheses.
 38. Implantable materials according to claim 37,characterised in that the prostheses are selected from the groupconsisting of cardiac valves, vessel prostheses, bone- and jointprostheses.
 39. Implantable materials according to claim 36,characterised in that the implantable materials are artificial and/orbiological carrier materials, which contain the dedifferentiated,programmable stem cells according to claims 14, 15, 21 or 22 or thetarget cells according to claims 33 to
 35. 40. Implantable materialsaccording to claim 39, characterised in that the carrier materials arebags or chambers for introduction into the human body.
 41. Use of bag orchamber according to claim 40, which contains islet cells according toclaim 33, for the production of a pharmaceutical construct for use as anartificial islet cell portchamber for the supply of insulin.
 42. Use ofa bag or chamber according to claim 40, which contains adipocytesaccording to claim 33, for the production of a pharmaceutical construct,which contains artificial polymers filled with adipocytes, for breastconstruction after surgery and for use in the case of plastic and/orcosmetic correction.
 43. Implantable materials according to claims 36 or40, characterised in that they are semi-permeable port chamber systems,which contain differentiated isolated somatic target cells according toclaim
 33. 44. Use of the semi-permeable port chamber system according toclaim 43 for the production of a pharmaceutical construct for in vivotreatment of endocrine, metabolic or haemostatic diseases.
 45. Use ofM-CSF and IL-3 for the production of dedifferentiated, programmable stemcells of human monocytic origin.